Science
Chemists synthesize active site of myoglobin Work on the muscle protein could lead to improved artificial lung machines and to increased ability of blood to carry oxygen Two chemists from the University of California, San Diego, have synthe sized the active site of the muscle pro tein myoglobin. The synthetic com pound exhibits essentially the same chemical properties as the natural sub stance. The man-made active site is com posed of the iron-containing heme por tion of a typical myoglobin molecule as well as a strategically placed imidazole grouping. Both create the physical and
chemical conditions that induce the synthetic molecule to bind oxygen reversibly "in approximately the same way" as the natural molecule does, ac cording to Dr. Teddy Traylor and grad uate student Chi Kuong Chang. Their finding indicates that the protein por tion of myoglobin is not as important to the active site as was formerly be lieved. Availability of the synthetic active site—or compounds similar to it— could lead to the development of im proved artificial lung machines or to substances that might increase the blood's ability to carry oxygen, the chemists suggest. Such compounds could also clarify what happens when oxygen binds to the active site of the myoglobin molecule, they add, noting that heretofore this has been impossi ble to determine in the intact mole cule.
Dr. Teddy Traylor adjusts molecular model of active site of myoglobin
Synthesis steps lead to myoglobin active site
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C&EN Nov. 5. 1973
Like the blood protein hemoglobin, the chief function of myoglobin is to transport oxygen. It is made up of a protein portion and a heme portion, the latter consisting of an iron atom bound to four nitrogen heterocycles. Myoglobin facilitates the transfer of oxygen from blood to muscle cells, which then store the oxygen as an energy source for muscular activity. In designing the synthetic active site, Dr. Traylor and Mr. Chang followed the earlier suggestion of Dr. J. H. Wang of Yale that the ability of myoglobin to bind oxygen and release it as needed may be partially due to the geometric configuration of an imidazole grouping relative to the iron atom in the heme portion of the molecule. Although the "protein scaffolding" appears to hold the imidazole in the correct position in the natural molecule, the objective of the California scientists was to achieve the same effect with simpler chemical groupings. Using available x-ray crystallographic data for myoglobin and observing computer-drawn stereoscopic representations of the active site region of the natural product, Dr. Traylor and Mr. Chang determined that a "handle" consisting of three methylene groups would hold the imidazole in the proper geometric configuration. The synthetic active site, known as ferropyrroporphyrin-iV[3-(l-imidazolyl)propyl]amide, was prepared in a series of steps beginning with chlorophyll a, a readily available component of spinach and other plants. The acid chloride of the intermediate pyrroporphyrin XV was reacted with l-(3-aminopropyl)imidazole to produce the amide. On reaction of the amide with ferrous sulfate/acetic acid and reduction with sodium dithionite, the imidazole group in the end product was held in the same geometric configuration it occupies in the naturally occurring site, say Dr. Traylor and Mr. Chang. In a series of experiments [Proc. Nat. Acad. Sci. USA, 70, 2647 (1973); and J. Amer. Chem. Soc, 95, 5810 (1973)], the two scientists demonstrated that the synthetic active site—like myoglobin—binds oxygen reversibly both in solution and in the solid state. And, like myoglobin and hemoglobin, it binds carbon monoxide much more strongly that it binds oxygen. When dissolved in a solution of methylene chloride at -45° C , the synthetic active site showed almost the same binding constant with oxygen as does sperm whale myoglobin at room temperature. The binding constant shows the ratio between bound and unbound oxygen. [In unrelated work, Dr. Jack Baldwin and his Massachusetts Institute of Technology coworkers devised a synthetic iron(II) complex that binds oxygen reversibly at -90° C. and decomposes above -50° C. (C&EN, Aug. 27, page 11)]. By comparing the visible spectrum of the product produced under the
proper conditions, the University of California scientists concluded that it did not resemble the starting material or the compound in which iron(II) is oxidized to iron(III). However, it strongly resembled the spectrum of oxygen-bound sperm whale myoglobin. Emphasizing the importance of the imidazole grouping in the functioning of the active site, Dr. Traylor and Mr. Chang found that when it was replaced by some other nitrogen heterocycle— for example, pyridine—the product did not bind oxygen but was oxidized under the same experimental conditions. They now attribute the unique binding power of myoglobin's active site both to the basic nature of the imidazole as well as to its geometric relationship to the iron(II) of the heme. Now that the myoglobin active site, stripped of the surrounding protein, has been made, Dr. Traylor and Mr. Chang believe that biochemists may be in a better position to resolve the 40year-old debate over whether the oxygen molecules bind to the heme in an end-to-end or sideways fashion. In addition, they propose that it may now be possible to study other biological catalysts using the same "stripped down" approach they applied to myoglobin. In a more practical sense, Dr. Traylor tells C&EN, it is possible that materials similar to the synthetic active
site could be built into plastic film to facilitate oxygen transport in artificial lung machines. In a typical lung machine, oxygen under pressure permeates from one side of a membrane to the side exposed to the blood of a patient undergoing lung surgery. Currently, the California chemists plan to study the effect of their compounds on the oxygen-transport properties of various polymers, following methods developed by Dr. W. J. Ward, III, of General Electric's research laboratories in Schenectady, N.Y. Dr. Traylor admits, however, that such compounds may not become really practical until they can bind oxygen at room temperature and not be oxidized. But he points out that the present synthetic active-site material already has been shown to be more stable in polystyrene than it is in solution. In addition, Dr. Traylor is confident that improved stability might be achieved by altering the basicity of the attached grouping, protecting the exposed portion of the heme with a hydrocarbon structure, or by some other as yet untried approach. Similarly, he believes that if a synthetic active site could be attached to serum albumin or some other biologically compatible protein, it should be possible to increase the oxygen-carrying capacity of blood in which hemoglobin is not doing the job it should.
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Lockheed Missiles 6C Space Company Nov. 5, 1973 C&EN
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